The purpose of the proposed research is to determine the detailed mechanism by which O2 oxidoreductases catalyze reactions of molecular oxygen. Since many O2 oxidoreductases are involved in defects of metabolism it is hoped that a greater understanding of their mechanisms will aid in the control or alleviation of such metabolic diseases. Three specific enzymes to be investigated during this grant period are: (1) galactose oxidase (the first enzyme for which evidence for trivalent copper as an intermediate has been obtained); (2) liver 4-hydroxyphenyl-pyruvate dioxygenase (absent or defective in various forms of tyrosinemia and responsible for the abnormal amounts of 0-hydroxyphenylacetic acid produced by persons with phenylketonuria); (3) kidney inositol oxygenase (which catalyzes the first irreversible step in mammalian catabolism of inositol, a required constituent of most animal cells). The methods to be used are those which have been proved so successful in arriving at a greater understanding of non-redox enzymic reactions. They will include: kinetic methods, isotope tracer studies, use of inactivators and modified substrate molecules, EPR techniques, etc.